The Effects Of ZEB1 On Activation And Transdifferentiation Of Rat Hepatic Stellate Cells

In this study, we use ELLSA, MTT and Western blotting to show mechanisms of TGF-βsignaling pathway in the activated hepatic stellate cells stimulated by acetaldehyde. The functions of the TGF-βpathways effects are also observed during hepatic stellate cell activation. Firstly, acetaldehyde is used to stimulate HSC cell for different time. MTT is applied to assay the proliferation level of activated HSC; ELISA is applied to detect the concentrations of fibronectin (FN) and TIMPⅠ. To confirm that acetaldehyde can stimulate the activation of hepatic stellate cells, SB-431542 is used to block TGF-βsignal pathway specifically, and Western blotting is applied to detect the expression of ZEB1,α-SMA, Smad3 and p-Smad3-S425 in the HSC, to show mechanisms of TGF-βsignal pathway. Methods:①Cells were divided into: (1) A control group cultured for 12h, (2) A control group cultured for 24h, (3) A control group cultured for 36h, (4) An experimental group with blocked TGF- beta signal pathway for 24h, (5) An experimental group with acetaldehyde stimulation and blocked TGF- beta signal pathway for 24h, (6) An experimental group with acetaldehyde stimulation for 12h, (7) An experimental group with acetaldehyde stimulation for 24h, (8) An experimental group with acetaldehyde stimulation for 36h. After the cells were cultured to 70%-80% confluences in 0.4% FBS DMEM medium to synchronize cells for 12h, the medium was replaced to 10% FBS DMEM complete medium. The inhibitors (SB-431542 final concentration is 10μmol / L) of the corresponding training were used to treat the experimental group with blocked TGF- beta signal pathway for 24h, experimental group with acetaldehyde stimulation and blocked TGF- beta signal pathway for 24h use the corresponding inhibitor (SB-431542 final concentration is 10μmol / L) pretreatment for 1h before the aldehyde stimulation (final concentration is 200μmol / L), time of additional stimulation is 12h,collect cells after the different stimulate times. Then the total cellular protein was extract, the protein content was detect by using G250, the total protein was separated by SDS-PAGE electrophoresis and the protein content was detected by Western blotting. to test targets including: The expression ofα-SMA, ZEB1,Smad3 and p-Smad3-S425 in the HSC. According to western blotting protein band intensity, Quantity One analysis software was used for processing.②HSC-T6 cells were also divided into: (1) A control group cutured for 12h, (2) A control group cutured for 24h, (3) A control group cutured for 36h, (4) An experimental group with blocked TGF- beta signal pathway for 24h, (5) An experimental group with acetaldehyde stimulation and blocked TGF- beta signal pathway for 24h, (6) An experimental group with acetaldehyde stimulation for 12h, (7) An experimental group with acetaldehyde stimulation for 24h, (8) An experimental group with acetaldehyde stimulation for 36h. Inoculate HSC-T6 cells beads in 96-well plates, each group with 3 holes. After the cells were cultured to 70%-80% confluences in 0.4% FBS DMEM medium to synchronize cells for 12h, the medium was replaced to 10% FBS DMEM complete medium. The inhibitors (SB-431542 final concentration is 10μmol / L) of the corresponding training were used to treat the experimental group with blocked TGF- beta signal pathway for 24h, experimental group with acetaldehyde stimulation and blocked TGF- beta signal pathway for 24h use the corresponding inhibitor (SB-431542 final concentration is 10μmol / L) pretreatment for 1h before the aldehyde stimulation (final concentration is 200μmol / L), time of additional stimulation is 12h. Then observe the growth conditions of cells under the light microscope, detect the activity of cells by MTT, using Elisa detect the content of collagenⅠand fibronectin FN. Results:①After being activated by acetaldehyde, the activation of HSC cells were significantly increased using MTT assay, collagenⅠand fibronectin FN content increase in different degrees using Elisa. It indicates the alcohol metabolite acetaldehyde stimulated significantly on the activation of hepatic stellate cells. After using the SB431542 blocked TGF-βpathway, the result of MTT showed the activation of HSC cells were significantly down-regulated, TIMPⅠand FN were also significantly down-regulated.②In the control groups, the expression ofα-SMA, ZEB1 and p-Smad3-S425 in the HSC were slightly increased. It explains that the control groups have basic activity; Comparing with control groups, activated groups were significantly up-regulated the expression ofα-SMA, ZEB1 and p-Smad3-S425 in the HSC; The groups of TGF-βblocked restrain the expression ofα-SMA, ZEB1 and p-Smad3-S425 obviously.③Through comparing the different stimulate times of HSC obtain the result that the expression of 24h groups as the highest,as comparing with 24h, 36h groups slightly decrease.Conclusion:①Acetaldehyde can activate HSC cells obviously, after the HSC cells is activated by acetaldehyde, HSC cells begin proliferation and transdifferentiation.②The effect of proliferation and transdifferentiation in HSC cells which have been activated by acetaldehyde can reach to the peak when the stimulating time is 24h.③Acetaldehyde can lead to activation of hepatic stellate cells and transdifferentiation by stimulating TGF-βsignaling pathway. Blocking TGF-βsignaling pathway by SB-431542 can restrain the acetaldehyde activate HSC cells effectively.④The activation of TGF-βsignaling pathway and the expression ofα-SMA and ZEB1 up-regulated significantly suggest that TGF-βis related to the EMT in human liver fibrosis.